Method for obtaining plants exhibiting enhanced resistance to water stress

ABSTRACT

A method for obtaining a plant having a modified amount of ASR protein, providing it with enhanced resistance to water stress compared to a non-transformed plant, comprises: transforming at least a plant cell with a vector, containing an expression cassette including a nucleotide sequence coding for an ASR protein or inhibiting the expression of an ASR protein; culturing the resulting transformed cell so as to generate a plant containing in its genome the expression cassette.

The present invention relates to a method for obtaining plantsexhibiting enhanced resistance to water stress.

In temperate regions, periods of low rainfall, which vary in intensityand are unpredictable, are the cause of a notable decrease inproductivity in crop plants. The water deficit can severely affect plantgrowth and reproduction. Various strategies making use of physiologicalmechanisms (decrease in growth of aerial parts, closure of stomata)and/or cellular mechanisms (osmotic adjustment) may enable them to evadethis water stress, or at the very least to tolerate it. Thesemechanisms, which are at least partly controlled by ABA (abscisic acid),a phytohormone the concentration of which increases in plants subjectedto water stress (Zeevaart and Creelman, 1988), involve many proteinswith different putative functions: proteins of the membrane channeltype, or proteins expressed in response to damage caused to the cell(proteases, protease inhibitors), or else proteins whose functions arenot directly related to stress but which are expressed at higher levelsunder conditions of water or salinity stress (enzymes of glycolysis, ofmethionine synthesis). The plant's response depends, moreover, onenvironmental (type of restraint, intensity, duration) and genetic(species and genotype) parameters, making it difficult to determine therole of these proteins in the mechanisms of tolerance to drought.

There is therefore a real need to demonstrate the function of candidategenes in the mechanisms of tolerance to water stress, in order to usethem in transgenesis aimed at obtaining plants exhibiting bettertolerance to water stress. This is particularly important for field cropspecies, such as maize, for example, which attains a level of maximumsensitivity to drought 15 days before and 15 days after flowering of theplant (July–August in Europe), a period during which the plant wouldabsorb 45% of its total needs.

A first study carried out by Riccardi et al. (1998) on a maize linedesignated Io (Iodent) has demonstrated about twenty proteins, theexpression of which is significantly increased in response to waterstress, and for which putative functions have been proposed.

One of these proteins, demonstrated in the Io line used by Riccardi etal. (1998) but not in the F2 line, exhibits homologies with a tomatoprotein which is induced by ABA, water stress and ripening of the fruit,ASR1 (ABA-water stress-ripening-induced protein), but the function ofwhich is still unknown (Iusem et al., 1993). Other genes showinghomologies with tomato ASR have been identified, in particular in potato(Silhavy et al., 1995), lemon (Canel et al., 1995) and rice (Thomas etal., 1999), but no function has been clearly demonstrated for theproteins encoded by these genes. A QTL (Quantitative Trait loci) studyhas made it possible to map the Asr1 gene in a region containing boththe locus controlling the amount of ASR1 and QTLs for foliar senescenceand protandry (De Vienne et al., 1999).

The authors of the present invention have now demonstrated the role of arecombinant ASR protein in the direct response of a plant to waterstress, in order to use transgenesis. Specifically, they have succeededin developing a method for obtaining a plant having a modified amount ofASR protein, conferring on it better resistance to water stress comparedto a nontransformed plant.

A subject of the invention is therefore a method for obtaining a plantcontaining a modified amount of ASR protein, conferring on it betterresistance to water stress compared to a nontransformed plant,comprising the steps consisting in:

-   -   transforming at least one plant cell with a vector containing an        expression cassette comprising a nucleotide sequence encoding an        ASR protein or blocking the expression of an ASR protein;    -   culturing the cell thus transformed so as to generate a plant        containing, in its genome, said expression cassette.

The term “ASR protein” is intended to mean a protein expressed naturallyby a plant in response to water stress, having an amino acid sequenceidentical or homologous to the maize sequence SEQ ID No 2.

The term “homologous sequence” is intended to mean preferably a sequenceexhibiting at least 50%, preferably 70%, similarity with the sequenceSEQ ID No 2.

For the purpose of the invention, included in the definition of “ASRprotein” are all the ASR proteins of various plants, such as, forexample, that of rice, and also the proteins which are modified, forexample by addition, deletion and/or substitution (preferablyconservative substitution) of a small number of amino acids.

Also included in the definition of “ASR protein” are the polypeptidesencoded by all or part of the sequence SEQ ID No 1, No 3, No 4 or No 5,or any homologous sequence, it being understood that these polypeptidesconserve the property of resistance to water stress.

The nucleic acid sequence encoding an ASR protein may more particularlybe a sequence from a cereal, in particular a sequence from maize.

Said sequence may advantageously be a cDNA sequence specific for thestate of water stress, isolated from a maize line by differentialhybridization. Such a sequence is given in the attached sequencelisting, and designated SEQ ID No 1. It is also possible to use agenomic DNA sequence encoding a maize ASR protein, as defined by one ofthe sequences SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5, or a sequencehomologous thereto, provided that it is expressed in modified amountscompared to the amount usually produced by a nontransformed plant.

In the attached sequence listing,

-   SEQ ID No 3 is a genomic DNA sequence isolated from a maize line    which strongly expresses the ASR protein,-   SEQ ID No 4 is a genomic DNA sequence isolated from a control maize    line A188, and-   SEQ ID No 5 is a genomic DNA sequence isolated from an F₂ maize    line.

Said sequence may in particular encode the amino acid sequence SEQ ID No2 of the maize ASR protein, or a variant thereof, for example a varianthaving the sequence SEQ ID No 2 with an insertion of a lysine residuebetween amino acids 55 and 56 of SEQ ID No 2.

It is also possible to use nucleotide sequences encoding ASRs from otherplants, such as, for example, those cited above.

The nucleic acid encoding an ASR protein is inserted into a nucleic acidconstruct, called expression cassette, and is functionally linked toelements which allow the expression thereof and, optionally, theregulation thereof.

Among these elements, mention may be made of promoters, activators andterminators of transcription.

Use may preferentially be made of a constitutive promoter, such as therice actin promoter, followed by the rice actin intron (RAP-RAI)contained in the plasmid pAct1-F4 (Mc Elroy et al., 1991) or the 35Spromoter (Kay et al., 1987), or a tissue-specific promoter. By way ofexample, mention may be made of the wheat HMWG promoter or the radishcruciferin gene promoter, PCRU, which both allow expression of theprotein of interest in the seeds (Anderson O. D. et al., 1989;Depigny-This et al., 1992). Use may advantageously be made of promotersequences which induce expression under water conditions (Kasuga et al.,1999). Among the terminators which can be used in the constructs of theinvention, mention may in particular be made of the 3′ end of theAgrobacterium tumefaciens nopaline synthase gene (Depicker et al.,1982). Mention may also be made of the 35S polyA terminator of thecauliflower mosaic virus (CaMV), described in the article by Franck etal. (1980).

The expression of the ASR protein can also be regulated by usingsequences such as peptide addressing signals (chloroplast addressingsignals, vacuolar addressing signals, addressing signals for endoplasmicretention, etc.), or such as intron sequences, enhancer sequences orleader sequences.

In the present invention, the sequence of interest may be a nucleotidesequence encoding an ASR protein, said sequence being placed in thesense direction, or a nucleotide sequence blocking the expression of anASR protein. This nucleotide sequence blocking the expression of the ASRprotein is preferentially a sequence encoding all or part of the ASRprotein, said sequence being placed in the antisense direction. When thesequence encoding an ASR protein is placed in the sense direction, atransformed plant is obtained which exhibits an increase in the ASRprotein compared to a nontransformed plant. This plant has the advantageof withstanding water stress more effectively than a nontransformedplant. When the sequence is placed in the antisense direction, atransformed plant is obtained which also exhibits a modification ofresistance to water stress. Without adhering in any way to a precisemechanism of action, this observation might be explained by the plantactivating an alternative pathway of resistance to water stress, inresponse to inhibition of the ASR by the antisense sequences.

The expression cassette is inserted into a nucleotide vector, such as aplasmid, which may also comprise a marker gene, for example a genemaking it possible to select between a transformed plant and a plantwhich does not contain the transfected foreign DNA. As marker gene,mention may be made of a gene which confers resistance to an antibiotic,for example to hygromycin (Herrera-Estrella et al., 1983) or resistanceto a herbicide such as the sulfonamide asulam (WO 98/49316).

This vector or any sequence encoding an ASR protein, such as thesequences SEQ ID No 1, SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5, orsequences homologous to the latter, can be used to transform plant cellsaccording to techniques commonly known to those skilled in the art, inorder to obtain plants exhibiting enhanced resistance to water stress.

According to one embodiment of the method of the invention, the plantcells are transformed with a vector as defined above, transferred into acellular host capable of infecting said plant cells by allowingintegration into the genome of the latter of the nucleotide sequences ofinterest initially contained in the genome of the abovementioned vector.Advantageously, the cellular host used is a bacterial strain, such asAgrobacterium tumefaciens, in particular according to the methoddescribed in the article by An et al. (1986), or else Agrobacteriumrhizogenes, in particular according to the method described in thearticle by Guerche et al. (1987).

For example, the plant cells can be transformed by transferring the Tregion of the Agrobacterium tumefaciens extrachromasomal, circular,tumor-indicating Ti plasmid, using a binary system (Watson et al.,1994). To do this, two vectors are constructed. In one of these vectors,the T region has been removed by deletion, with the exception of theleft and right borders, a marker gene being inserted between them so asto allow selection in the plant cells. The other partner of the binarysystem is a helper Ti plasmid, which is a modified plasmid which nolonger has a T region but which still contains the vir virulence genesrequired for transformation of the plant cell.

According to a preferred mode, use may be made of the method describedby Ishida et al. (1996), for the transformation of monocotyledons.

Other embodiments of the method of the invention may also be mentioned,in particular methods of direct gene transfer into plant cells, such asdirect microinjection into plant embryoids (Neuhaus et al., 1987),infiltration under vacuum (Bechtold et al., 1993) or electroporation(Chupeau et al., 1989), or else direct precipitation using PEG (Schocheret al., 1986) or bombardment with particles covered with the plasmid DNAof interest, using a particle gun (M. Fromm et al., 1990).

According to another protocol, the transformation is carried outaccording to the method described by Finer et al. (1992), using atungsten or gold particle gun.

The subject of the invention is also a host cell transformed with thenucleic acid sequences described above, and also a plant or part of aplant, in particular fruit, seed, grain, pollen, leaf or tuber, whichcan be obtained using one of the methods set out above.

They may, for example, be field crop plants (wheat, rapeseed, sunflower,peas, soybean, barley, in particular maize, etc.) or vegetables andflowers.

The hybrid transgenic plants, obtained by crossing at least one plantaccording to the invention with another, are also part of the invention.

The invention in particular relates to a plant exhibiting an increase inexpression of the ASR protein compared to a nontransformed plant, forexample a 2- to 3-fold increase. This increase in expression of the ASRprotein confers enhanced resistance to water stress on the transformedplants.

The resistance to water stress of the transformed plants according tothe invention, compared to the control plants, can be assessed usingvarious morphological, physiological and/or biochemical measuringmethods, for particular irrigation conditions. By way of example, thetolerance to stress can be measured by phenotypic observation (i) offoliar senescence, by morphological measurements and by assaying thechlorophyll in the foliar disks, (ii) of the protandry or date offlowering of the male and female plants, (iii) of the growth of theplant, by measuring the final length and width of the leaves and alsothe final height of the plant, and by studying the rolling up of theleaves, or else (iv) of the yield of grain, of the weight of a thousandgrains and of the number of ears per plant.

The stress experienced by the plants can also be evaluated by measuringthe ABA content (method of Quarrie et al., 1988) or by measuring thewater potential, or else, where appropriate, by monitoring expression ofthe protein by two-dimensional electrophoresis using a leaf sample.

The plants obtained according to the invention can also be used inallele complementation experiments in order to validate the function ofthe inserted gene. Use of the transformants in backcross experimentsmakes it possible to introduce only the gene inserted in the parentalgenetic background, without other sequences which might influence thephenotype of the recombinant with regard to tolerance to drought.

Preferably, the inserted gene is coupled with a selectable marker gene,which facilitates the monitoring of the backcrosses and, consequently,the monitoring of the insertion of the gene of interest into the line inwhich it is desired to validate the effect.

The principle consists in crossing the transformant with the parentalline not possessing the favorable allele of the gene of interest, andcomparing the phenotypes of the recombinant line with the parentallines. It is also possible to use transformants containing the genomicsequences in these complementation experiments. This complementationassay makes it possible to verify in particular that overexpression ofthe cDNA in the sense direction complements the effect of the weak (ornull) allele.

Thus, it is, for example, possible to verify that the ASR1 gene is thegene responsible for the QTL and PQL (protein quantitative loci), foundat this genetic position on chromosome 10 in maize. Since the amount ofthis protein varies between different lines, stronger expressions; oreven expressions of more favorable alleles, can be detected andexploited for improving plants. Plants exhibiting favorable alleles canbe detected by immunoassays with antibodies specific for the ASR1protein (ELISA assay, Western blotting, etc.).

The use of a nucleic acid encoding an ASR, of a fragment of this nucleicacid, as a probe or primer for PCR-type amplification, in order toselect transformed plants exhibiting better resistance to water stress,also falls within the context of the invention.

The nucleic acid sequences encoding an ASR, such as those designated SEQID No 1, SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5, and also anyoligonucleotide obtained from one of these sequences, can thus be usedas probes in marker-assisted selection programs, for example forfollowing the introgression of the gene encoding the maize ASR proteininto a plant. For this, at least one of these probes is labeled, forexample with a radioactive isotope, and then brought into contact withgenomic DNA from the plant, predigested with restriction enzymes, underconditions which allow specific hydration of the labeled probe to theDNA in question. Other techniques using for example PCR may also be usedto carry out genotyping.

The following figures and examples illustrate the invention withoutlimiting the scope thereof.

LEGEND TO THE FIGURES

FIG. 1 represents a restriction map of the plasmid pWP 280 containingthe promoter pActin intron-barnase-Nos PolyA.

FIG. 2 represents a restriction map of the intermediate vector pBIOS 308containing ZmASR1 cDNA in the sense direction.

FIG. 3 represents a restriction map of the intermediate vector PBIOS 309containing ZMASR1 cDNA in the antisense direction.

FIG. 4 represents the effect of each “antisense” or “sense”transformation event, relative to its own control (Basta sensitive), onfoliar senescence, the effect being measured on the first day of gradingafter flowering.

FIG. 5 represents kinetics of the effect, for all the “sense”,“nontransformed” and “antisense” events, possibly placed underconditions of water stress, on foliar senescence.

EXAMPLES Example 1

Obtaining and Cloning the cDNA of ZmAsr1 from a Maize Line WhichStrongly Expresses the ASR Protein

a) Culture Conditions and Taking of Samples for the Plants Having BeenUsed to Isolate the cDNA

The authors of the invention cloned the ZmAsr1 cDNA from Io maize lines(Riccardi et al., 1998) or from maize lines selected according to thesame criteria as Io.

The maize plants are grown in perlite under controlled conditions in aculturing chamber (illumination: 450 mmol m⁻² s⁻¹, photoperiod: 16 h,day/night temperature: 25° C./20° C., relative humidity 60%), wateredwith a nutrient solution. When the plants have reached the “5 leaf”stage (5th emerged leaf), the watering is either stopped, for the plantsunder conditions of water stress, or it is continued, for the controlplants. Ten days later, samples are taken from the ensheathed part ofthe blade of the 7th leaf.

b) Isolating the cDNAs Specific for the State of Water Stress byDifferential Hybridization

A cDNA library is prepared from mRNA from the leaves of stressed plantsusing the Lambda ZapII cDNA synthesis/Gigapack GoldI cloning kit(Stratagene, La Jolla, USA), according to the manufacturer'sintructions.

The mRNAs prepared from the leaves of stressed plants and of controlplants are transcribed into radiolabeled cDNA using 100 μCi of ³²P-dATPand hexanucleotides serving as random primers (Sambrook et al., 1989).The single-stranded cDNAs, originating either from the stressed plant orfrom the control plant, are hybridized, in the same proportion, on thecDNA clones of the library. The hybridization temperature in thephosphate buffer/SDS/EDTA system (Church and Gilbert, 1984) is 68° C.and the final washes are carried out with solutions containing 0.1×SSC,0.05% (weight/volume) SDS.

The labeled clones are recovered by in vivo excision of the phagemidaccording to the Stratagene protocol using E. coli SOLR and the“Exassist” helper phage. The DNA of these clones is sequenced andcompared to nucleic acid sequence libraries (BLAST) according to themethod described in Altschul et al. (1990). One of these clones exhibitsstrong homology with tomato Asr1 and is named ZmAsr1 for Zea maize Asr1.

Example 2

ZmASR1 Genomic Sequences

The primers cASR1-1F (5′-TGTCGATCCAATTGTCACTT-3′)=SEQ ID No 6 andcASR1-740R (5′-TGGAGAAACGTAAACAACTA-3′)=SEQ ID No 7, defined at the twoends of the cDNA sequence of the ASR1 protein, are used in PCRamplification on maize line total DNA. The PCR reactions are carried outaccording to conventional techniques.

The PCR products are analyzed by electrophoresis: a band at 900 bp isrecovered in order to extract the DNA therefrom and clone it accordingto conventional techniques. After verification of their size, theinserts are extracted and sequenced.

Example 3

Construction of Chimeric Genes for the Constitutive Expression of theASR1 Protein or Else of an Antisense Leading to Inhibition Thereof.

First, 2 basic plasmid vectors are constructed, pBIOS 306 and pBIOS 307,containing the actin promoter-actin intron (pAct), the cDNA of the Asr1gene, respectively in the sense and antisense direction, and thenopaline synthase terminator (terNos) which introduces a polyadenylationsignal which is functional in many plant species.

Intermediate vectors are then produced for homologous recombination withthe Japan Tobacco vector pSB1 (EP 672 752) in Agrobacterium tumefaciensstrain LBA 4404 (Hoekema et al., 1983).

The transfer followed by the expression of the genes (gene for selectionand gene of interest) into maize is based on the natural properties ofAgrobacterium tumefaciens (Zambrisky et al., 1989) and on thesuperbinary plasmid strategy (Hiei et al., 1994 and Ishida et al.,1996).

The restriction enzymes used for the cloning are provided by New EnglandBiolabs (New England Biolabs, UK). The enzymatic reactions are carriedout by following the protocols described, by Sambrook et al., in themolecular cloning manual (Molecular Cloning: A Laboratory Manual, 1989,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

a) Construction of the Basic Plasmid Vectors for the ConstitutiveExpression of the Asr1 Gene and of its Antisense

The molecular constructs for the constitutive expression of the Asr1gene in the sense and antisense direction were prepared as describedbelow:

-   -   cloning of the 795 bp I/XhoI fragment (Asr1 cDNA=SEQ ID No 1)        into the EcoRV-restricted vector pBIOS 298.

The vector pBIOS 298 contains the actin promoter-actin intron (pAct) (McElroy et al., 1991) and the Nos terminator. This vector was generated bydeletion of the 366 bp PstI fragment (Barstar gene) of the vector pWP280, containing the pActin-intron˜Barstar˜Nos polyA cassette (FIG. 1).

This nonoriented cloning makes it possible to obtain 2 new vectors:

-   -   the vector pBIOS 306 carrying the gene pAct-ZmAsr1 sense-terNos    -   the vector pBIOS 307 carrying the gene pAct-ZmAsr1        antisense-terNos.

The orientation of the cDNA relative to the actin promoter wasdetermined by simple enzymatic restriction with EagI and doubleenzymatic restriction with Hind III-EcoRV.

b) Construction of the Intermediate Vectors for Homologous Recombinationwith pSB1 (Obtaining Superbinary Plasmids)

The vectors used for the homologous recombination in Agrobacteriumtumefaciens are derived from the vector pBIOS 273.

Construction of the Plasmid pBIOS 273

The basic vector for the homologous recombination is the vector PBIOS273. This vector was generated in 2 steps:

-   -   Cloning of the BspDI/XhoI fragment (pAct-Bar-terNos) of the        vector pDM 302 (Cao et al., 1992) into the SmaI and BspDI sites        of the vector pSB12 (Japan Tobacco). The vector resulting from        this cloning is called pBIOS 272.    -   Deletion of the XhoI site at position 3363 of the vector pBIOS        272 by partial digestion with XhoI and the action of DNA        Polymerase I large (Klenow) fragment. The vector obtained, which        has a unique XhoI site, is named pBIOS 273.

Generation of the Intermediate Recombination Vectors Containing the Asr1cDNA in the Sense or Antisense Direction

These constructs were generated from the vector pBIOS 274, derived fromthe vector pBIOS 273 by cloning the XhoI fragment(ProA9-Barnase-terCaMV) of the vector pWP 128 (Paul et al., 1992) intothe XhoI-restricted vector pBIOS 273.

The intermediate vectors pBIOS 308 and pBIOS 309 were obtained bycloning the 2970 bp SalI/XhoI fragments of the Vectors pBIOS 306 andpBIOS 307 into the BspDI/XhoI sites of the vector PBIOS 274.

This cloning thus makes it possible to substitute thepA9-Barnase-terCaMV gene of the vector pBIOS 274 with the pAct-ZmAsr1sense-terNos gene, the resulting vector being called pBIOS 308 (FIG. 2),or with the pAct-ZmAsr1 antisense-terNos gene, the resulting vectorbeing called pBIOS 309 (FIG. 3).

c) Construction of the Superbinary Transformation Vectors for Expressionof the Asr1 Gene and of Its Antisense in Agrobacterium tumefaciens andin Maize Plants

Construction of the Superbinary Vectors

The vectors used for the transformation of maize are derived fromhomologous recombination of the plasmids pBIOS 308 and pBIOS 309 withthe vector pSB1 (EP 672 752). The vector pSB1 contains the virB and virGgenes of the Ti plasmid pTiBo542 present in the Agrobacteriumtumefaciens strain A281 (ATCC 37349), the tetracyclin resistance gene,an origin of replication functional in E. coli and Agrobacterium, and ahomologous region found in the intermediate vectors pBIOS 308 and pBIOS309. The presence of this homologous region in the recipient plasmid(pSB1) and the intermediate plasmids (pBIOS 308 and PBIOS 309) isresponsible for the phenomenon of homologous recombination.

The intermediate vectors pBIOS 308 and pBIOS 309 are introduced into theAgrobacterium tumefaciens cells containing the vector pSB1 byelectroporation using the GIBCO BRL CELL PORATOR Voltage Boosteraccording to the method described by Mattanovitch et al. (1989) and theprotocol provided by the supplier (Life Technologies, USA).

The agrobacteria containing the superbinary vectors are selected on YTCaCl₂ medium in the presence of rifampicin and spectinomycin at aconcentration of 50 mg/l. The rifampicin resistance gene is carried outby the bacterial chromosome. The spectinomycin resistance, carried bythe plasmids pBIOS 308 and pBIOS 309 (origin of replication in E. coli),may be expressed only after homologous recombination with the vectorpSB1 (origin of replication functional in Agrobacterium and E. coli).

The superbinary plasmids obtained after recombination are named pRec 308(PBIOS 308×pSB1) and pRec 309 (pBIOS 309×pSB1). They possess origins ofreplication which are functional both in E. coli and in Agrobacteriumtumefaciens, the tetracyclin resistant and the spectinomycin resistantgenes, the T-DNA in which are located the cassettes of expression of theBar and Asr1 (sense or antisense cDNA) genes, and the virB and virGvirulence genes of the plasmid pTiBo542.

Characterization of the Superbinary Vectors pRec 308 and pRec 309

These superbinary plasmids pRec 308 (Asr1 gene in the sense direction)and pRec 309 (Asr1 gene in the antisense direction) are characterized byenzymatic restriction with SalI. Southern blotting analysis of the SalIrestriction fragments is then carried out with the Bar probe and theAsr1 gene probe (this probe corresponds to the 795 bp EcoRI/XhoIfragment of the vector pHHU516, therefore to the complete cDNA). Theprofiles obtained are those expected.

Example 4

Maize Plant Transformation

The maize plant transformation is carried out according to the protocolof Ishida et al. (1996).

The transformation begins with a co-culture in which the immatureembryos of the maize plants (size ranging from 1 to 1.2 mm) are broughtinto contact, for 5 minutes, with Agrobacterium tumefaciens LBA 4404containing the superbinary vectors pRec 308 or pRec 309. The embryos arethen placed on LSAs medium for 3 days in the dark and at 25° C.

The following step is that of the first selection of the transformedcalluses: the “embryo-calluses” are transferred onto LSD 5 mediumcontaining phosphinotricine at 5 mg/l and cefotaxime at 250 mg/l(elimination of Agrobacterium tumefaciens). This step is carried out 2weeks in the dark and at 25° C.

The second selection step is carried out by transferring the embryoswhich have developed on LSD 5 medium onto LSD 10 medium(phosphinotricine at 10 mg/l) in the presence of cefotaxime, for 3 weeksunder the same conditions as in the first selection (25° C., in thedark).

The third selection step consists in excising the type I calluses(fragments of 1 to 2 mm) and in transferring them into the dark for 3weeks at 25° C. on LSD 10 medium in the presence of cefotaxime.

The regeneration of the plantlets is then carried out by excising thetype I calluses which have proliferated and transferring them onto LSZmedium in the presence of phosphinotricine at 5 mg/l and cefotaxime, fortwo weeks at 22° C. and under continuous light. The plantlets which haveregenerated are transferred onto rooting medium (Ishida et al., 1996)for two weeks at 22° C. and under continuous illumination for thedevelopment step.

The plants obtained are then transferred to the phytotron for thepurpose of acclimatizing them.

Example 5

Demonstration of Expression of the ZMASR1 Protein in the TransformedPlants

The proteins of the leaf samples originating from the transformed plantswere extracted according to the method of Damerval et al. (1986), andwere analyzed by two-dimensional electrophoresis (TDE) according to theprotocol of Riccardi et al. (1998).

Two-dimensional electrophoresis consists in separating polypeptides as afunction of their isoelectric point and as a function of their molecularweight (electrophoresis in the presence of SDS). Before electrophoresis,the proteins are extracted and kept under denaturing conditions: thequaternary structure is eliminated. The various polypeptides making upthe oligomeric proteins migrate independently during the twoelectrophoreses. The gels are then stained with silver nitrate.

The two-dimensional gel thus obtained is compared with that producedusing proteins of nontransformed A188 plant leaves.

The results obtained from the plants transformed with the codingsequence placed in the “sense” direction show, by simple visualexamination of these two gels, stronger expression of the ASR1 proteinin the transformed plants compared to the A188 control plants.

The ASR spot observed on the plants transformed with the antisenseconstruct appear to be still detectable, but less strongly, which showsthat the “antisense” transformants express the protein less than thecontrol plants.

This protein analysis therefore demonstrates expression of the ASR1protein in the transformed plants, in an amount which is modifiedcompared to the nontransformed plants.

Example 6

Measurement of the Tolerance to Water Stress of the Transgenic PlantsObtained According to the Invention

The resistance to water stress of the transformed plants according tothe invention, compared with the control plants, can be assessed usingvarious phenotypic, physiological and/or biochemical analytical methods,for particular irrigation conditions under normal conditions(conventional culture with watering) and under conditions of waterstress.

By way of example, the water conditions in the field may be as follows:

-   -   normal irrigation in the irrigated part on the basis of 5 mm per        day controlled by tensiometers placed 30, 50 and 70 cm deep;    -   restrictive irrigation, with no supply of water, if possible,        until 10 days after flowering. At this approximate date, it is        decided to provide water when the stress is judged to be too        intense; the rhythm of supply should not exceed 3 mm per day.

The meteorological data and the irrigation conditions are recorded.

The grading carried out at the various periods of flowering and ofharvesting consist in measuring:

a) Measurement of the Tolerance to Stress by Phenotypic Observation

The genetic analyses carried out beforehand suggested a potential rolefor ASR1 in foliar senescence and protandry (time difference betweenmale and female flowering) under conditions of drought. Thesecharacteristics are therefore preferentially studied.

Foliar senescence can be studied with morphological measurements whichconsist in counting the number of dried up leaves and green leaves at 4dates 15 days apart, from the date of flowering, or at various stages ofdevelopment. When very different behaviors are observed concerning therolling up and the color of the leaves, an assessment is made accordingto a scale of 0 to 5 (from the most tolerant to the least tolerant tostress).

Senescence can also be measured by assaying chlorophyll on samples offoliar disks from the ear leaf at flowering.

Protandry, or time difference between the dates of flowering of the maleplants (presence of pollen) and female plants (bristles coming out) ismeasured as follows: the dates of appearance of the bristles and of thepollen are noted individually for each plant, and the dynamics arerepresented on a curve of percentage of plants having flowered as afunction of time.

Moreover, various assessments were carried out at harvest in order toevaluate the effect of the tolerance to stress on grain production, inparticular: the percentage fertilization (ratio of the number of grainsper ear/number of fertilizable ovules), the number of rows per ear, thenumber of grains per row, the water content of the grains, the weight of1000 grains and the number of fusarium infected plants.

The use of a nondestructive identification test using basta makes itpossible to easily distinguish the plants which are resistant to bastafrom the sensitive plants, in each transgenic descendance, the resistantplants containing the Asr gene genetically linked to the selectablemarker gene, unless there has been a recombination event. This testconsists in swabbing the end of a leaf with a solution of basta andobserving the resulting phenotype, without the vitality of the wholeplant being threatened: the end of the leaf undergoes necrosis and driesout when the plant is sensitive, or remains green if the plant isresistant. This makes it possible to observe, on the measurements oftolerance to stress, whether the ASR transgene positively influences theresponse to stress as a function of expression of the gene in the sensedirection or in the antisense direction.

b) Evaluation of the Stress Experienced by the Plants

Samples are taken from the leaves, in order to measure the ABA content(method of Quarrie et al., 1988) and, where appropriate, the expressionof the protein by two-dimensional electrophoresis.

The stress experienced by the plants can be evaluated by measuring thebasic water potential using a portable pressure chamber onnontransformed plants, under control conditions and under conditions ofwater stress. Measurements of the relative water content of the leavescan also be made.

Foliar senescence, which permits a decrease in the surface ofevaporation, and re-mobilization of metabolites to the remainder of theplant, was measured as described above, on adult plants after flowering,by counting the number of leaves of which at least 50% of the surface isdry.

A significant effect of expression of the ASR protein on the proportionof dry leaves is observed. In fact, when related to their own controls,the “sense” events show, at a given time, a greater proportion of dryleaves than “antisense” events (FIG. 4). Similarly, under conditions ofwater stress, the foliar senescence kinetics measurements show that the“sense” events become senescent more rapidly than the “nontransformed”and that the “antisense” events become senescent less rapidly than the“nontransformed” (FIG. 5).

The term “sense” event is intended to mean an event derived from aninitial step of transformation with an expression cassette containingthe ASR sequence placed in the sense direction. The term “antisense”event is intended to mean an event derived from an initial step oftransformation with a cassette containing the ASR sequence placed in theantisense direction.

The “sense” events, which become senescent more rapidly than the“nontransformed” under conditions of water stress, therefore exhibit aselective advantage of tolerance to water stress, in particular in thecase of a stress of long duration (decrease in the surface ofevaporation and re-mobilization of metabolites to the remainder of theplant).

The “antisense” events, which become senescent less rapidly than the“nontransformed” under conditions of water stress, also exhibit anadvantage, in particular in the case of a water deficit of shortduration. In fact, these plants will have maintained a greater surfaceof evaporation and will therefore benefit more fully from a subsequentsupply of water (rain or irrigation).

In addition, the measurements of the stress experienced by the plants(ABA content in leaves) revealed a slight but highly significant stress:respectively 616 and 512 ng ABA/g solids in the stressed and controlplants, i.e. an increase of approximately 100 ng of ABA per gram ofsolids in the stressed plants. This response is the same for the “sense”and “antisense” events, and also for the nontransformed plants. Thetransformation does not therefore appear to have an effect on theaccumulation of ABA in the leaves.

These results therefore confirm that, in the presence of a slight waterstress and of low ASR expression in the transformed plants, significantdifferences are already observed.

Moreover, the effect of the tolerance to water stress on grainproduction was measured relative to the yield of grain, the weight ofone thousand grains and the number of ears per plant. The resultsobtained with a low water stress show grain yield measurementscomparable between transformed (“sense” and “antisense”) plants andnontransformed plants, taken under conditions of stress or under normalconditions.

The transformation, firstly, and the tolerance to water stress,secondly, do not therefore appear to affect the grain yield of theplants.

Plant growth measurements were also taken. The lengths of 3 leaves aboveand below the ear were measured on “sense” and “antisense” plants afterflowering, in the absence of water stress. A highly significantdifference was observed for the 3 leaves:

for the leaves

-   -   F0, antisense=78.41 cm (p<0.01)        -   sense=76.11 cm    -   F1, antisense=77.96 cm (p<0.01)        -   sense=75.76 cm    -   F2, antisense=75.04 cm (p<0.01)        -   sense=72.94 cm

Overall, a significant difference of 2 cm in length is thereforeobserved for these 3 leaves, the leaves of the “sense” plants beingsmaller than those of the “antisense” plants. This decrease in leafgrowth observed in the “sense” events therefore correlates with agreater senescence, the two phenomena resulting in a decreased surfaceof evaporation, which allows the plant to more successfully toleratewater stress.

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1. A method for obtaining a transformed plant exhibiting an increasedamount of ASR (ABA-water stress-ripening-induced) protein, conferring onsaid plant increased resistance to water deficit stress compared to anon-transformed plant, comprising transforming at least one plant cellwith a vector containing an expression cassette comprising a nucleotidesequence encoding an ASR protein, wherein said ASR protein comprises theamino acid sequence of SEQ ID NO: 2; and culturing the cell thustransformed so as to generate a plant containing, in its genome, saidexpression cassette, and wherein said plant expresses said protein. 2.The method according to claim 1, wherein said nucleotide sequenceencoding said ASR protein comprises sequence SEQ ID No
 1. 3. The methodaccording to claim 1, wherein the expression cassette comprises apromoter for constitutive expression of the nucleotide sequence encodingthe ASR protein.
 4. A plant, or plant part comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 2, wherein saidplant or plant part is obtained by the method of claim
 1. 5. The plant,or part of a plant, according to claim 4, that exhibits an increase inexpression of the ASR protein compared to a non-transformed plant. 6.The plant, or part of a plant, according to claim 4, which is a fieldcrop plant selected from maize, wheat, rapeseed, sunflower and peas. 7.The plant, or part of a plant, according to claim 4, which is maize. 8.The method according to claim 3, wherein said promoter is a rice actinpromoter operably linked to a rice actin gene intron.